Up to now, there has been proposed a method of radiating excitation light to an object disposed on a surface of a substrate to observe the shape of the object. For example, Patent Literature 1 describes an apparatus which radiates excitation light outputted from an excitation light source to a transparent substrate, causes total reflection of the excitation light inside of the transparent substrate to thereby generate an evanescent wave on a substrate surface, and detects scattered light of the evanescent wave due to a sample on the substrate. Note that the apparatus described in Patent Literature 1 does not disperse the scattered light.
In addition, for example, Patent Literature 2 describes an apparatus which disperses fluorescence and scattered light deriving from sample components excited by an evanescent wave. Note that, in the apparatus described in Patent Literature 2, the sample components are not captured on a flow path boundary plane.
Meanwhile, there has been proposed an apparatus which captures a plurality of biological molecules on a substrate surface, generates an evanescent wave in a given range of the substrate surface similarly to Patent Literature 1, and creates an image of light emitted from the biological molecules excited by the evanescent wave. Nonfluorescent biological molecules are captured on the substrate, a reaction liquid containing fluorescent molecules is fed onto the substrate, and fluorescence from a capture position of the biological molecules is observed. This enables observation of a binding reaction between the biological molecules and the molecules contained in the reaction liquid. For example, first, unmodified single-stranded DNA is captured on the substrate, a reaction liquid containing fluorescently-modified bases modified with fluorescent materials different for each base type is introduced, and fluorescence from a molecule capture position is dispersed while complementary bases are bound to the single-stranded DNA, whereby the sequence of the captured DNA can be decoded.
In recent years, as described in Non Patent Literature 2, it has been proposed to capture DNA and the like on a substrate and determine a nucleotide sequence thereof. Sample DNA fragments to be analyzed are randomly captured molecule by molecule on the substrate surface to be elongated substantially for each base, and results thereof are detected by fluorescence measurement, whereby a nucleotide sequence thereof is determined. Specifically, in one cycle, performed are: a step of causing a DNA polymerase reaction using four types of dNTP derivatives (MdNTPs) which can be each taken into a template DNA as a substrate of a DNA polymerase to stop a DNA chain elongation reaction by the presence of a protecting group, the dNTP derivatives each having a detectable label; then, a step of detecting the taken-in MdNTPs by fluorescence detection or the like; and a step of bringing the MdNTPs back into an elongatable state. Such a cycle of the steps is repeated to determine the nucleotide sequence of the sample DNA. According to this technique, because the sequences of the DNA fragments can be determined molecule by molecule, a large number of fragments can be analyzed at the same time, leading to enhancement in analysis throughput. In addition, according to this technique, because there is a possibility that the nucleotide sequence can be determined for each single DNA molecule, the need for purification and amplification of a sample DNA in cloning or PCR, which has been a problem in conventional techniques, may be eliminated, and an increase in speed of genomic analysis and genetic diagnosis can be expected. Note that, according to this technique, because the sample DNA fragment molecules to be analyzed are randomly captured on the substrate surface, an expensive camera having a pixel count of several hundred times the number of the captured DNA fragment molecules is necessary. That is, in the case where an interval between the DNA fragment molecules is adjusted to 1 micrometer on average, an interval between some molecules is larger, and an interval between other molecules is smaller. Hence, in order to detect such molecules separately from one another, it is necessary to detect a fluorescent image at a smaller interval in terms of the substrate surface. In general, measurement at an interval of about one tenth is necessary.
Meanwhile, in Non Patent Literature 3 and Patent Literature 3, the sensitivity of fluorescence detection is further enhanced according to a nano-aperture evanescent radiation/detection method which can further reduce the radiation volume of excitation light than the total-reflection evanescent radiation/detection method. Two glass substrates, that is, a glass substrate A and a glass substrate B are disposed parallel to each other, and a planar aluminum thin film, which includes a nano-aperture having a diameter of 50 nm and has a film thickness of about 100 nm, is laminated on a surface of the glass substrate A, the surface facing the glass substrate B. The aluminum thin film needs to have a light-blocking property. A reaction vessel is formed between the two glass substrates, and the reaction vessel is filled with a solution, whereby a solution layer is formed between the two glass substrates. The reaction vessel is provided with an inlet and an outlet for the solution. The solution is fed from the inlet, and the solution is discharged from the outlet, whereby the solution can be flowed in a direction parallel to the glass substrates and the aluminum thin film. This enables the solution in the solution layer to have given compositions. If excitation light which is oscillated from an Ar ion laser and has a wavelength of 488 nm is radiated perpendicularly to the glass substrate A from the opposite side to the glass substrate B while being focused by an objective lens, an evanescent field of the excitation light is formed in a portion of the solution layer near a bottom plane inside of the nano-aperture, and the excitation light does not further propagate outside of the portion of the solution layer. Meanwhile, fluorescence emission is detected by forming an image thereof on a two-dimensional CCD using the objective lens. In the evanescent field, the excitation light intensity exponentially decreases with increasing distance from the nano-aperture bottom plane, and the excitation light intensity is 1/10 at a distance of approximately 30 nm from the nano-aperture bottom plane. Further, according to the nano-aperture evanescent radiation/detection method, the radiation width of excitation light in the direction parallel to the glass substrates is limited to the aperture diameter, that is, 50 nm, unlike the total-reflection evanescent radiation/detection method, and hence the radiation volume of excitation light is further reduced. For this reason, background light caused by, for example, fluorescence emission of free fluorescent materials and Raman scattering of water can be dramatically reduced. As a result, only fluorescent materials labeled to target biological molecules can be selectively detected in the presence of higher-concentration free fluorescent materials, so that extremely high-sensitive fluorescence detection can be achieved. In these literatures, such a fluorescence detection method as described above is applied to take-in measurement of dNTPs using an elongation reaction of DNA molecules.